U.S. patent application number 14/124370 was filed with the patent office on 2014-07-17 for therapeutic compounds for immunomodulation.
This patent application is currently assigned to Aurigene Discovery Technologies Limited. The applicant listed for this patent is Muralidhara Ramachandra, Pottayil G. N. Sasikumar, K. Rajeev Shrimali, Krishnaprasad Subbarao, Suresh Kumar Vadlamani. Invention is credited to Muralidhara Ramachandra, Pottayil G. N. Sasikumar, K. Rajeev Shrimali, Krishnaprasad Subbarao, Suresh Kumar Vadlamani.
Application Number | 20140199334 14/124370 |
Document ID | / |
Family ID | 47295571 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140199334 |
Kind Code |
A1 |
Sasikumar; Pottayil G. N. ;
et al. |
July 17, 2014 |
Therapeutic Compounds for Immunomodulation
Abstract
The present invention provides Immunosuppressive compounds
capable of inhibiting the programmed cell death 1 (PD1) signalling
pathway. The present invention further provides peptide based
compositions for treatment of cancer or treatment of infections via
immunopotentiation caused by inhibition of immunosuppressive
signalling induced by PD-1, PD-L1, or PD-L2 and therapies using
them, immunopotentiative substrates included as the active
ingredient. Further, the invention provides pharmaceutical
compositions comprising the Immunosuppressive peptide compounds or
modified peptide moieties for preventive and/or therapeutic agents
for cancer, cancer metastasis, immunodeficiency, an infectious
disease or the like and an application of PD-1 or PD-L1 as a
testing or diagnostic agent or a research agent for such a
disease.
Inventors: |
Sasikumar; Pottayil G. N.;
(Bangalore, IN) ; Ramachandra; Muralidhara;
(Bangalore, IN) ; Vadlamani; Suresh Kumar;
(Bangalore, IN) ; Shrimali; K. Rajeev; (Hyderabad,
IN) ; Subbarao; Krishnaprasad; (Bangalore,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sasikumar; Pottayil G. N.
Ramachandra; Muralidhara
Vadlamani; Suresh Kumar
Shrimali; K. Rajeev
Subbarao; Krishnaprasad |
Bangalore
Bangalore
Bangalore
Hyderabad
Bangalore |
|
IN
IN
IN
IN
IN |
|
|
Assignee: |
Aurigene Discovery Technologies
Limited
Bangalore
IN
|
Family ID: |
47295571 |
Appl. No.: |
14/124370 |
Filed: |
December 21, 2011 |
PCT Filed: |
December 21, 2011 |
PCT NO: |
PCT/IN2011/000881 |
371 Date: |
December 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61515007 |
Aug 4, 2011 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
530/323 |
Current CPC
Class: |
Y02A 50/414 20180101;
Y02A 50/467 20180101; Y02A 50/491 20180101; Y02A 50/481 20180101;
Y02A 50/471 20180101; Y02A 50/473 20180101; C07K 5/0227 20130101;
C07K 7/02 20130101; Y02A 50/385 20180101; Y02A 50/409 20180101;
Y02A 50/30 20180101; Y02A 50/478 20180101; A61K 38/00 20130101;
Y02A 50/402 20180101; Y02A 50/463 20180101; Y02A 50/406 20180101;
Y02A 50/465 20180101; Y02A 50/469 20180101; Y02A 50/411 20180101;
Y02A 50/401 20180101; A61K 38/08 20130101 |
Class at
Publication: |
424/185.1 ;
530/323 |
International
Class: |
C07K 7/02 20060101
C07K007/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 8, 2011 |
IN |
19438/CHE/2011 |
Claims
1. A peptide derivative of formula (I), ##STR00062## wherein,
Am.sub.1 represents 1 to 4 amino acid residues which may be same or
different and each independently selected from Ser, Asn and Thr;
wherein one of the peptide bond (--CONH--) between any two amino
acid residues may be replaced with a modified peptide bond of
##STR00063## wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl; Am.sub.2 is comprising of dipeptide
selected from Ser-Phe or Phe-Ser, wherein Phe may be optionally
substituted with amino(C.sub.1-C.sub.20)alkyl, --NHCOCH.sub.3 or
--NHCONH.sub.2; X is Glu which may optionally form amide bonds with
its alpha carboxylic acid group, delta carboxylic acid group or
amino group; L is a linker selected from --NH(CH.sub.2).sub.nNH--,
--NH(CH.sub.2).sub.nCH(NH.sub.2)CO--, --OOC(CH.sub.2).sub.mCOO--,
--NH(CH.sub.2).sub.nCO--, --NH(CH.sub.2CH.sub.2O).sub.nNH--,
--NH(CH.sub.2CH.sub.2O).sub.nCO-- or
--CO(CH.sub.2CH.sub.2O).sub.nCO--; R.sub.1 is free C-terminal,
amidated C-terminal or N-terminal of Am.sub.1; or is
(C.sub.1-C.sub.20)acyl substitution; R.sub.2 is free C-terminal,
amidated C-terminal or N-terminal of Am.sub.2; or Y--R.sub.5; Y is
an optional linker selected from --OOC(CH.sub.2).sub.mCOO--,
--CO(CH.sub.2).sub.nNH--, --CO(CH.sub.2CH.sub.2O).sub.nNH-- or
--COCH.sub.2(OCH.sub.2CH.sub.2).sub.nNH--; R.sub.5 is an albumin
binding moiety such as maleimido propionic acid; R.sub.3 is free
alpha C-terminal, amidated alpha C-terminal or N-terminal of Glu;
`n` is an integer selected from 2 to 10, both inclusive; `m` is an
integer selected from 0 to 8, both inclusive; wherein one or more
or all amino acids may be in a D-configuration; or its retro
analogue or a pharmaceutically acceptable stereoisomer or a
pharmaceutically acceptable salt thereof.
2-12. (canceled)
13. A peptide derivative of claim 1 having the formula (Ia):
##STR00064## wherein, R.sub.1 is N-terminal of Ser; or
(C.sub.1-C.sub.20)acyl substituted with either hydroxyl group or
amino group of Ser L is a linker selected from
--NH(CH.sub.2).sub.nNH--, --NH(CH.sub.2).sub.nCH(NH.sub.2)CO--,
--OOC(CH.sub.2).sub.mCOO--, --NH(CH.sub.2).sub.nCO--,
--NH(CH.sub.2CH.sub.2O).sub.nNH--,
--NH(CH.sub.2CH.sub.2O).sub.nCO-- or
--CO(CH.sub.2CH.sub.2O).sub.nCO--; R.sub.2 is free C-terminal,
amidated C-terminal or N-terminal of Am.sub.2; or Y--R.sub.5; Y is
an optional linker selected from --OOC(CH.sub.2).sub.mCOO--,
--CO(CH.sub.2).sub.nNH--, --CO(CH.sub.2CH.sub.2O).sub.nNH-- or
--COCH.sub.2(OCH.sub.2CH.sub.2).sub.nNH--; R.sub.5 is an albumin
binding moiety such as maleimido propionic acid; R.sub.3 is OH or
NH.sub.2; R.sub.4 is a substituent on phenyl group of Phe and is
selected from hydrogen, amino(C.sub.1-C.sub.20)alkyl,
--NHCOCH.sub.3 or --NHCONH.sub.2; `n` is an integer having values
selected from 2 to 10, both inclusive; `m` is an integer having
values selected from 0 to 8, both inclusive; and one of the peptide
bond (--CONH--) of Ser-Asn, Asn-Thr or Thr-Ser may be replaced with
a modified peptide bond of ##STR00065## wherein Q is hydrogen,
--CO(C.sub.1-C.sub.20)alkyl or --COO(C.sub.1-C.sub.20)alkyl group;
wherein one or more or all amino acids may be in the
D-configuration; or retro analogue or a pharmaceutically acceptable
stereoisomer or a pharmaceutically acceptable salt thereof.
14. The compound according to claim 13, wherein one or more or all
amino acids are in the D-configuration.
15. The compound according to claim 13, wherein L is
--NH(CH.sub.2).sub.4NH--.
16. The compound according to claim 13, wherein L is
--NH(CH.sub.2).sub.4CH(NH.sub.2)CO--.
17. The compound according to claim 13, wherein R.sub.2 is
N-terminal of Ser.
18. The compound according to claim 13, wherein the peptide bond
(--CONH--) of Ser-Asn is replaced with a modified peptide bond of
##STR00066## wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl group.
19. The compound according to claim 13, wherein the peptide bond
(--CONH--) of Asn-Thr is replaced with a modified peptide bond of
##STR00067## wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl group.
20. A peptide derivative of claim 1 having the formula (Ib):
##STR00068## wherein, R.sub.1 is free C-terminal or amidated
C-terminal of Ser; L is a linker selected from
--NH(CH.sub.2).sub.nNH-- or --NH(CH.sub.2CH.sub.2O).sub.nNH--;
R.sub.4 is selected from hydrogen, amino(C.sub.1-C.sub.20)alkyl,
--NHCOCH.sub.3 or --NHCONH.sub.2; wherein one or more or all amino
acids may be in D-configuration; or retro analogue or a
pharmaceutically acceptable stereoisomer or a pharmaceutically
acceptable salt thereof.
21. The compound according to claim 20, wherein one or more or all
amino acids are in D-configuration.
22. The compound according to claim 20, wherein L is
--NH(CH.sub.2).sub.4NH--.
23. The compound according to claim 20, wherein R.sub.1 is amidated
C-terminal of Ser.
24. A compound selected from TABLE-US-00006 Comp No. Structure 001
##STR00069## (SEQ ID NO: 3) 002 ##STR00070## (SEQ ID NO: 4) 003
##STR00071## (SEQ ID NO: 5) 004 ##STR00072## (SEQ ID NO: 6) 005
##STR00073## (SEQ ID NO: 7) 006 ##STR00074## (SEQ ID NO: 8) 007
##STR00075## (SEQ ID NO: 9) 008 ##STR00076## (SEQ ID NO: 10) 009
##STR00077## (SEQ ID NO: 11) 010 ##STR00078## *D-Phe (SEQ ID NO:
12) 011 ##STR00079## (SEQ ID NO: 13) 012 ##STR00080## *D-Glu (SEQ
ID NO: 14) 013 ##STR00081## *D-Phe urea (SEQ ID NO: 15) 014
##STR00082## (SEQ ID NO: 16) 015 ##STR00083## *D-Glu (SEQ ID NO:
17) 016 ##STR00084## *All D-amino acids (SEQ ID NO: 18) 017
##STR00085## (SEQ ID NO: 19) 018 ##STR00086## (SEQ ID NO: 20) 019
##STR00087## (SEQ ID NO: 21) 020 ##STR00088## (SEQ ID NO: 22) 021
##STR00089## (SEQ ID NO: 23) 022 ##STR00090## (SEQ ID NO: 24) 023
##STR00091## (SEQ ID NO: 25) 024 ##STR00092## (SEQ ID NO: 26) 025
##STR00093## (SEQ ID NO: 27) 026 ##STR00094## (SEQ ID NO: 28) 027
##STR00095## (SEQ ID NO: 29) 028 ##STR00096## (SEQ ID NO: 30) 029
##STR00097## (SEQ ID NO: 31) 030 ##STR00098## (SEQ ID NO: 32) 031
##STR00099## (SEQ ID NO: 33)
25. A compound according to claim 1, for use as a medicament for
the treatment of cancer or infectious disease.
26. A pharmaceutical composition comprising a compound according to
claim 1, and a pharmaceutically acceptable diluent or carrier.
27. (canceled)
28. A method of inhibiting growth of tumour cells and/or metastasis
in a subject, comprising administering to the subject a
therapeutically effective amount of compound according to claim 1,
capable of inhibiting the programmed cell death 1 (PD1) signaling
pathway.
29. The method of claim 28, wherein the tumour cells are of a
cancer selected from the group consisting of melanoma, renal
cancer, prostate cancer, breast cancer, colon cancer and lung
cancer.
30. (canceled)
31. A method of treating an infectious disease in a subject
comprising administering to the subject a therapeutically effective
amount of compound according to claim 1, capable of inhibiting the
programmed cell death 1 (PD1) signaling pathway such that the
subject is treated for the infectious disease
32. A method of treating bacterial and viral infections in a
subject comprising administering to the subject a therapeutically
effective amount of compound according to claim 1, capable of
inhibiting the programmed cell death 1 (PD1) signaling pathway such
that the subject is treated for the bacterial, fungal and viral
infections.
33. (canceled)
34. A method for treating sepsis in a subject comprising
administering to the subject a therapeutically effective amount of
compound according to claim 1, capable of inhibiting the programmed
cell death 1 (PD1) signaling pathway such that the subject is
treated for the bacterial, fungal and viral infections.
Description
RELATED APPLICATION
[0001] This application claims the benefit of Indian provisional
application number 1943/CHE/2011 filed on Jun. 8, 2011 and U.S.
provisional application No. 61/515,007 filed on Aug. 4, 2011 all of
which hereby incorporated by reference.
FIELD OF INVENTION
[0002] The present invention relates to novel peptides as
therapeutic agents capable of inhibiting the programmed cell death
1 (PD1) signalling pathway.
[0003] The invention also relates to modifications and derivatives
of the therapeutic agents.
[0004] The invention further relates to pharmaceutical compositions
comprising the said novel peptides and their derivatives as
therapeutic agents.
[0005] The invention also encompasses the use of the said
therapeutic agents, modifications and derivatives for treatment of
disorders via immunopotentiation comprising inhibition of
immunosuppressive signal induced due to PD-1, PD-L1, or PD-L2 and
therapies using them.
BACKGROUND OF THE INVENTION
[0006] Programmed Cell Death 1 or PD-1 (also referred to as PDCD1)
is a .about.55 kD type I membrane glycoprotein (Shinohara T et al,
Genomics, 1994, Vol. 23, No. 3, 704-706). PD-1 is a receptor of the
CD28 superfamily that negatively regulates T cell antigen receptor
signalling by interacting with the specific ligands and is
suggested to play a role in the maintenance of self tolerance.
[0007] PD-1 peptide relates to almost every aspect of immune
responses including autoimmunity, tumour immunity, infectious
immunity, transplantation immunity, allergy and immunological
privilege.
[0008] The PD-1 protein's structure comprise of-- [0009] an
extracellular IgV domain followed by [0010] a transmembrane region
and [0011] an intracellular tail
[0012] The intracellular tail contains two phosphorylation sites
located in an immunoreceptor tyrosine-based inhibitory motif and an
immunoreceptor tyrosine-based switch motif, which suggests that
PD-1 negatively regulates TCR signals. Also, PD-1 is expressed on
the surface of activated T cells, B cells, and macrophages, (Y.
Agata et al., Int Immunol, May 1996, 8, 765.) suggesting that
compared to CTLA-4 [(Cytotoxic T-Lymphocyte Antigen 4), also known
as CD152 (Cluster of differentiation 152) is a protein that also
plays an important regulatory role in the immune system], PD-1 more
broadly negatively regulates immune responses.
[0013] PD-1 has two ligands, PD-L1 (Programmed Death Ligand 1 or
PDCD1L1 or B7-H1) (Freeman G J et al, Journal of Experimental
Medicine, 2000, Vol. 19, No. 7, 1027-1034.) and PD-L2 (Programmed
Death Ligand 2 or PDCD1L2 or B7-DC) (Latchman Y et al, Nature
Immunology, 2001, Vol. 2, No. 3, 261-267.), which are members of
the B7 family. PD-L1 is known to be expressed not only in immune
cells, but also in certain kinds of tumour cell lines (such as
monocytic leukaemia-derived cell lines, mast cell tumour-derived
cell lines, hematoma-derived cell lines, neuroblastoma-derived cell
lines, and various mammary tumour-derived cell lines) and in cancer
cells derived from diverse human cancer tissues (Latchman Y et al,
Nature Immunology, 2001, Vol. 2, No. 3, 261-267.) and on almost all
murine tumour cell lines, including PA1 myeloma, P815 mastocytoma,
and B16 melanoma upon treatment with IFN-.gamma. (Y. Iwai et al.,
Proc Natl Acad Sci USA, Sep. 17, 2002, 99, 12293. and C. Blank et
al., Cancer Res, February 2004, 64, 1140.). Similarly PD-L2
expression is more restricted and is expressed mainly by dendritic
cells and a few tumour cell lines. PD-L2 expression has been
verified in Hodgkin's lymphoma cell lines and others. There is a
hypothesis that some of the cancer or tumour cells take advantage
from interaction between PD-1 and PD-L1 or PD-L2, for suppressing
or intercepting T-cell immune responses to their own (Iwai Y et al,
Proceedings of the National Academy of Science of the United States
of America, 2002, Vol. 99, No. 19, 12293-12297.).
[0014] Tumour cells and virus (including HCV and HIV) infected
cells are known to express the ligand for PD-1 (to create
Immunosuppression) in order to escape immune surveillance by host T
cells. It has been reported that the PD-1 gene is one of genes
responsible for autoimmune diseases like systemic lupus
erythematosus (Prokunina et al, Nature Genetics, 2002, Vol. 32, No.
4, 666-669.). It has also been indicated that PD-1 serves as a
regulatory factor for the onset of autoimmune diseases,
particularly for peripheral self-tolerance, on the ground that
PD-1-deficient mice develop lupus autoimmune diseases, such as
glomerulonephritis and arthritis (Nishimura H et al, International
Immunology, 1998, Vol. 10, No. 10, 1563-1572; Nishimura H et al,
Immunity, 1999, Vol. 11, No. 2, 141-151.), and dilated
cardiomyopathy-like disease (Nishimura H et al, Science, 2001, Vol.
291, No. 5502, 319-332.).
[0015] Hence, in one approach, blocking the interaction of PD-1
with its ligand (PD-L1, PD-L2 or both) may provide an effective way
for specific tumour and viral immunotherapy.
[0016] Wood et al in U.S. Pat. No. 6,808,710 discloses method for
downmodulating an immune response comprising contacting an immune
cell expressing PD-1 with an antibody that binds to PD-1, in
multivalent form, such that a negative signal is transduced via
PD-1 to thereby down modulate the immune response. Such an antibody
may be a cross-linked antibody to PD-1 or an immobilized antibody
to PD-1.
[0017] Freeman et al in U.S. Pat. No. 6,936,704 and its divisional
patent U.S. Pat. No. 7,038,013 discloses isolated nucleic acids
molecules, designated B7-4 nucleic acid molecules, which encode
novel B7-4 polypeptides, isolated B7-4 proteins, fusion proteins,
antigenic peptides and anti-B7-4 antibodies, which co-stimulates T
cell proliferation in vitro when the polypeptide is present on a
first surface and an antigen or a polyclonal activator that
transmits an activating signal via the T-cell receptor is present
on a second, different surface.
[0018] There are some reports regarding substances inhibiting
immunosuppressive activity of PD-1, promoting or inhibiting the
interaction between PD-1 and PD-L1 or PD-L2, as well as the uses
thereof. PD-1, PD-L1 or PD-L2 inhibitory antibody, nucleic acid
molecules or polypeptides are reported in W0200114557,
WO2004004771, WO2004056875, WO2002079499, WO2003042402,
WO2002086083, WO2001039722, WO2003042402 and W0200200730.
[0019] WO2007005874 describes isolated human monoclonal antibodies
that specifically bind to PD-L1 with high affinity. The disclosure
provides methods for treating various diseases including cancer
using anti-PD-L1 antibodies.
[0020] US20090305950 describes multimers, particularly tetramers of
an extracellular domain of PD-1 or PD-L1. U.S. Pat. No. 7,432,059
and U.S. Pat. No. 7,709,214 disclose isolated nucleic acids
molecules, designated PD L2 nucleic acid molecules which encode
novel B7 related molecules which are ligands for PD 1.
[0021] Despite existence of many disclosures as discussed above,
however, a significant unmet medical need still exists due to the
lack of effective peptides or modified peptides as therapeutic
agents as alternatives in the therapeutic area. It is known that
synthetic peptides offer certain advantages over antibodies such as
ease of production with newer technologies, better purity and lack
of contamination by cellular materials, low immunogenicity,
improved potency and specificity. Peptides may be more stable and
offer better storage properties than antibodies. Moreover, often
peptides possess better tissue penetration in comparison with
antibodies, which could result in better efficacy. Peptides can
also offer definite advantages over small molecule therapeutics
counterparts such as lesser degree of toxicity and lower
probability of drug-drug interaction.
[0022] The present invention therefore may provide the solution for
this unmet medical need by offering novel synthetic peptide and its
derivatives which are based on the PD1 ectodomain.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0023] FIG. 1 shows the effect of compounds on mouse splenocyte
proliferation inhibited by PD1-PDL1 interaction in the presence of
human breast carcinoma cell line, MDA-MB-231 over expressing
PDL1.
AMINO ACID SEQUENCE INFORMATION
[0024] SEQ ID NO: 1 shows amino acid sequence of extracellular
domain of human PD-1.
[0025] SEQ ID NO: 2 shows amino acid sequence of BC Loop.
SUMMARY OF INVENTION
[0026] In accordance with the present invention, novel modified
peptides are provided which are capable of suppressing and/or
inhibiting the programmed cell death 1 (PD1) signalling
pathway.
[0027] In one aspect, the invention provides a modified peptide of
formula (I):
##STR00001##
[0028] wherein, Am.sub.1 represents 1 to 4 amino acid residues
which may be same or different and each independently selected from
Ser, Asn and Thr; wherein one of the peptide bond (--CONH--)
between any two amino acid residues may be replaced with a modified
peptide bond of
##STR00002##
[0029] wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl;
[0030] Am.sub.2 is comprising of dipeptide selected from Ser-Phe or
Phe-Ser, wherein Phe may be optionally substituted with
amino(C.sub.1-C.sub.20)alkyl, --NHCOCH.sub.3 or --NHCONH.sub.2;
[0031] X is Glu which may optionally form amide bonds with its
alpha carboxylic acid group, delta carboxylic acid group or amino
group;
[0032] L is a linker selected from --NH(CH.sub.2)--NH--,
--NH(CH.sub.2).sub.nCH(NH.sub.2)CO--, --OOC(CH.sub.2).sub.mCOO--,
--NH(CH.sub.2)--CO--, --NH(CH.sub.2CH.sub.2O).sub.nNH--,
--NH(CH.sub.2CH.sub.2O).sub.nCO-- or
--CO(CH.sub.2CH.sub.2O).sub.nCO--;
[0033] R.sub.1 is free C-terminal, amidated C-terminal or
N-terminal of Am.sub.1; or is (C.sub.1-C.sub.20)acyl
substitution;
[0034] R.sub.2 is free C-terminal, amidated C-terminal or
N-terminal of Am.sub.2; or Y--R.sub.5;
[0035] Y is an optional linker selected from
--OOC(CH.sub.2).sub.mCOO--, --CO(CH.sub.2)--NH--,
--CO(CH.sub.2CH.sub.2O).sub.nNH-- or
--COCH.sub.2(OCH.sub.2CH.sub.2)--NH--;
[0036] R.sub.5 is an albumin binding moiety such as maleimido
propionic acid;
[0037] R.sub.3 is free alpha C-terminal, amidated alpha C-terminal
or N-terminal of Glu;
[0038] `n` is an integer selected from 2 to 10, both inclusive;
[0039] `m` is an integer selected from 0 to 8, both inclusive; or
its retro analogue or a pharmaceutically acceptable stereoisomer or
a pharmaceutically acceptable salt thereof.
[0040] In another aspect, the invention provides a modified peptide
of formula (Ia):
##STR00003##
[0041] wherein,
[0042] R.sub.1 is N-terminal of Ser; or (C.sub.1-C.sub.20)acyl
substituted with either hydroxyl group or amino group of Ser
[0043] L is a linker selected from --NH(CH.sub.2)--NH--,
--NH(CH.sub.2).sub.nCH(NH.sub.2)CO--, --OOC(CH.sub.2).sub.mCOO--,
--NH(CH.sub.2)--CO--, --NH(CH.sub.2CH.sub.2O).sub.nNH--,
--NH(CH.sub.2CH.sub.2O).sub.nCO-- or
--CO(CH.sub.2CH.sub.2O).sub.nCO--;
[0044] R.sub.2 is free C-terminal, amidated C-terminal or
N-terminal of Am.sub.2; or Y--R.sub.5;
[0045] Y is an optional linker selected from
--OOC(CH.sub.2).sub.mCOO--, --CO(CH.sub.2)--NH--,
--CO(CH.sub.2CH.sub.2O).sub.nNH-- or
--COCH.sub.2(OCH.sub.2CH.sub.2)--NH--;
[0046] R.sub.5 is an albumin binding moiety such as maleimido
propionic acid;
[0047] R.sub.3 is OH or NH.sub.2;
[0048] R.sub.4 is a substituent on phenyl group of Phe and is
selected from hydrogen, amino(C.sub.1-C.sub.20)alkyl,
--NHCOCH.sub.3 or --NHCONH.sub.2;
[0049] `n` is an integer having values selected from 2 to 10, both
inclusive;
[0050] `m` is an integer having values selected from 0 to 8, both
inclusive; and
[0051] one of the peptide bond (--CONH--) of Ser-Asn, Asn-Thr or
Thr-Ser may be replaced with a modified peptide bond of
##STR00004##
[0052] wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl group; or retro analogue or a
pharmaceutically acceptable stereoisomer or a pharmaceutically
acceptable salt thereof.
[0053] In yet another aspect, the invention provides a modified
peptide of formula (Ib):
##STR00005##
[0054] wherein,
[0055] R.sub.1 is free C-terminal or amidated C-terminal of
Ser;
[0056] L is a linker selected from --NH(CH.sub.2).sub.nNH-- or
--NH(CH.sub.2CH.sub.2O).sub.nNH--;
[0057] R.sub.4 is selected from hydrogen,
amino(C.sub.1-C.sub.20)alkyl, --NHCOCH.sub.3 or --NHCONH.sub.2; or
retro analogue or a pharmaceutically acceptable stereoisomer or a
pharmaceutically acceptable salt thereof
DETAILED DESCRIPTION OF THE INVENTION
[0058] The term `peptide` is used herein to designate a sequence of
natural or unnatural amino acids bonded in said sequence by peptide
bonds.
[0059] The term `compound(s)` as used herein comprises peptides and
modified peptides as disclosed in the present invention.
[0060] The following common abbreviations of the amino acids are
used throughout this specification:
TABLE-US-00001 Gly (or G)--glycine Ala (or A)--alanine Val (or
V)--valine Leu (or L)--leucine Ile (or I)--isoleucine
Orn--ornithine Pro (or P)--proline Phe (or F)--phenylalanine Trp
(or W)--tryptophan Met (or M)--methionine Ser (or S)--serine Thr
(or T)--threonine Cys (or C)--cysteine Tyr (or Y)--tyrosine Asn (or
N)--asparagine Gln (or Q)--glutamine Asp (or D)--aspartic acid Glu
(or E)--glutamic acid Lys (or K)--lysine Arg (or R)--arginine His
(or H)--histidine
[0061] The term "(C.sub.1-C.sub.20)alkyl" as used herein refers to
straight or branched chain hydrocarbon having 1 to 20 carbon atoms
including, but not limited to, methyl, ethyl, propyl, butyl,
isobutyl and the like.
[0062] The term "amino(C.sub.1-C.sub.20)alkyl" as used herein
refers to straight or branched chain hydrocarbon having 1 to 20
carbon atoms with an amino group including, but not limited to,
aminomethyl, aminoethyl and the like.
[0063] The term "(C.sub.1-C.sub.20)acyl" as used herein refers to
RC(O)--, wherein R is (C.sub.1-C.sub.20)alkyl as defined above. For
example, acetyl, --C(O)(CH.sub.2).sub.4CH.sub.3,
--C(O)(CH.sub.2).sub.14CH.sub.3 and the like.
[0064] The term "peptide bond" as used herein refers to the
chemical bond between carbon and nitrogen in the bivalent group
CONH that unites amino acid residues in a peptide.
[0065] The term "retro analogue" as used herein refers to a
sequence of amino acids that has been altered with respect to a
native amino acid sequence by the reversal of the direction of the
native amino acid sequence. For example, for a native sequence
"Ser-Asn-Thr-Ser"; the retro sequence would be "Ser-Thr-Asn-Ser".
The retro analogue may be partial retro sequence.
[0066] The term "partial retro sequence" as used herein refers to a
sequence of amino acids that has been partially altered with
respect to a native amino acid sequence by the reversal of the
direction of the native amino acid sequence. For example, for a
native sequence "Ser-Asn-Thr-Ser-Glu-Phe-Ser"; the partial retro
sequence would be "Phe-Ser-Glu-Ser-Thr-Asn-Ser".
[0067] The term "albumin binding moiety" as used herein refers to
the moiety capable of binding to a serum albumin of a mammal which
may alternatively, be an organic, non-proteinaceous compound with
affinity for the mammalian serum albumin. The moiety is preferably
a radical of such an organic compound, which is covalently bound to
the biologically active protein moiety. For example, maleimido
propionic acid, maleimidocaproic acid hydrazide (commonly referred
to as EMCH) and the like.
[0068] The present invention provides immunosuppression modulating
peptides capable of suppressing and/or inhibiting the programmed
cell death 1 (PD1) signalling pathway.
[0069] The present invention further provides modifications,
derivatives of the peptides and pharmaceutical compositions
comprising the peptides for treatment of cancer or infection via
immunopotentiation caused by inhibition of immunosuppressive signal
induced by PD-1, PD-L1, or PD-L2 and therapies using them,
immunopotentiative substrates included as the active
ingredients.
[0070] In accordance with the present invention, in one of the
embodiment there are provided compounds capable of inhibiting
ability to inhibit the programmed cell death 1 (PD1) signalling
pathway and being capable of reducing PD-L1 or PD-L2 binding to
PD-1 and resulting immunosuppressive signalling by PD-1, wherein
the compound comprises a peptide moiety or a modified peptide
moiety.
[0071] The complete amino acid sequence of human PD-1 is disclosed
in U.S. Pat. No. 5,629,204 (Honjo et. al.) and Finger et al.,
(Gene, 1997, 197, 177-187.). Human and mouse PD-1 share around 60%
amino acid identity, whereas the extracellular IgV domain shows
only 21% and 16% sequence identity with CD28 and CTLA4,
respectively.
[0072] PD-1 possesses an ectodomain having multiple loop structures
and strands between the loops. The amino acid sequence of the human
PD-1 ectodomain is as set forth in SEQ ID NO: 1.
TABLE-US-00002 Extracellular domain of human PD-1 SEQ ID NO: 1
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAF
PEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPK
AQIKESLRAELRVTERRAEVPTAHPSPSPRSAGQFQTLV
[0073] These loop and strand assignments of amino acids are based
on the 1.8-.ANG.-resolution structure of the murine PD-1/PD-L2
complex reported in Lazar-Molnar et al, (PNAS, 2008, 105, 30,
10483-10488.).
[0074] Out of the various loops and strands of the PD-1 ectodomain,
BC loop (i.e. 24.sup.th to 30.sup.th amino acid of SEQ ID NO: 1)
was taken up for further modification. The present invention
provides compounds comprising of modified BC loop of extracellular
domain of human PD-1.
TABLE-US-00003 BC Loop SEQ ID NO: 2 SNTSESF
[0075] Modifications of the peptides discussed herein where
relevant include replacements of some or all of the L-amino acids
by D-amino acids, bonding of amino acids at other than alpha amino
groups, including at side chain amino or carboxylic groups,
inclusion of non-peptide linkers between peptide sequences,
deletion of one or more amino acids, cross-linking, lipidation,
stapling, and PEGylation.
[0076] The compounds of the invention may comprise linear or
branched peptides.
[0077] In one aspect, the invention provides a modified peptide of
formula (I):
##STR00006##
[0078] wherein, Am.sub.1 represents 1 to 4 amino acid residues
which may be same or different and each independently selected from
Ser, Asn and Thr; wherein one of the peptide bond (--CONH--)
between any two amino acid residues may be replaced with a modified
peptide bond of
##STR00007##
[0079] wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl;
[0080] Am.sub.2 is comprising of dipeptide selected from Ser-Phe or
Phe-Ser, wherein Phe may be optionally substituted with
amino(C.sub.1-C.sub.20)alkyl, --NHCOCH.sub.3 or --NHCONH.sub.2;
[0081] X is Glu which may optionally form amide bonds with its
alpha carboxylic acid group, delta carboxylic acid group or amino
group;
[0082] L is a linker selected from --NH(CH.sub.2)--NH--,
--NH(CH.sub.2).sub.nCH(NH.sub.2)CO--, --OOC(CH.sub.2).sub.mCOO--,
--NH(CH.sub.2)--CO--, --NH(CH.sub.2CH.sub.2O).sub.nNH--,
--NH(CH.sub.2CH.sub.2O).sub.nCO-- or
--CO(CH.sub.2CH.sub.2O).sub.nCO--;
[0083] R.sub.1 is free C-terminal, amidated C-terminal or
N-terminal of Am.sub.1; or is (C.sub.1-C.sub.20)acyl
substitution;
[0084] R.sub.2 is free C-terminal, amidated C-terminal or
N-terminal of Am.sub.2; or Y--R.sub.5;
[0085] Y is an optional linker selected from
--OOC(CH.sub.2).sub.mCOO--, --CO(CH.sub.2)--NH--,
--CO(CH.sub.2CH.sub.2O).sub.nNH-- or
--COCH.sub.2(OCH.sub.2CH.sub.2)--NH--;
[0086] R.sub.5 is an albumin binding moiety such as maleimido
propionic acid;
[0087] R.sub.3 is free alpha C-terminal, amidated alpha C-terminal
or N-terminal of Glu;
[0088] `n` is an integer selected from 2 to 10, both inclusive;
[0089] `m` is an integer selected from 0 to 8, both inclusive;
or its retro analogue or a pharmaceutically acceptable stereoisomer
or a pharmaceutically acceptable salt thereof
[0090] In another aspect, the invention provides a modified peptide
of formula (Ia):
##STR00008##
[0091] wherein,
[0092] R.sub.1 is N-terminal of Ser; or (C.sub.1-C.sub.20)acyl
substituted with either hydroxyl group or amino group of Ser
[0093] L is a linker selected from --NH(CH.sub.2)--NH--,
--NH(CH.sub.2).sub.nCH(NH.sub.2)CO--, --OOC(CH.sub.2).sub.mCOO--,
--NH(CH.sub.2)--CO--, --NH(CH.sub.2CH.sub.2O).sub.nNH--,
--NH(CH.sub.2CH.sub.2O).sub.nCO-- or
--CO(CH.sub.2CH.sub.2O).sub.nCO--;
[0094] R.sub.2 is free C-terminal, amidated C-terminal or
N-terminal of Am.sub.2; or Y--R.sub.5;
[0095] Y is an optional linker selected from
--OOC(CH.sub.2).sub.mCOO--, --CO(CH.sub.2)--NH--,
--CO(CH.sub.2CH.sub.2O).sub.nNH-- or
--COCH.sub.2(OCH.sub.2CH.sub.2)--NH--;
[0096] R.sub.5 is an albumin binding moiety such as maleimido
propionic acid;
[0097] R.sub.3 is OH or NH.sub.2;
[0098] R.sub.4 is a substituent on phenyl group of Phe and is
selected from hydrogen, amino(C.sub.1-C.sub.20)alkyl,
--NHCOCH.sub.3 or --NHCONH.sub.2;
[0099] `n` is an integer having values selected from 2 to 10, both
inclusive;
[0100] `m` is an integer having values selected from 0 to 8, both
inclusive; and
[0101] one of the peptide bond (--CONH--) of Ser-Asn, Asn-Thr or
Thr-Ser may be replaced with a modified peptide bond of
##STR00009##
[0102] wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl group;
or retro analogue or a pharmaceutically acceptable stereoisomer or
a pharmaceutically acceptable salt thereof
[0103] In yet another aspect, the invention provides a modified
peptide of formula (Ib):
##STR00010##
[0104] wherein,
[0105] R.sub.1 is free C-terminal or amidated C-terminal of
Ser;
[0106] L is a linker selected from --NH(CH.sub.2).sub.nNH-- or
--NH(CH.sub.2CH.sub.2O).sub.nNH--;
[0107] R.sub.4 is selected from hydrogen,
amino(C.sub.1-C.sub.20)alkyl, --NHCOCH.sub.3 or --NHCONH.sub.2; or
retro analogue or a pharmaceutically acceptable stereoisomer or a
pharmaceutically acceptable salt thereof
[0108] The embodiment below are illustrative of the present
invention and are not intended to limit the claims to the specific
embodiments exemplified.
[0109] According to one embodiment, specifically provided are
compounds of the formula (Ia) in which R.sub.1 is N-terminal of
Am.sub.1.
[0110] According to another embodiment, specifically provided are
compounds of the formula (Ia) in which R.sub.1 is
(C.sub.1-C.sub.20)acyl.
[0111] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which L is
--NH(CH.sub.2).sub.nNH-- and `n` is 4.
[0112] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which L is
--NH(CH.sub.2).sub.nCH(NH.sub.2)CO-- and `n` is 4.
[0113] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.2 is N-terminal of
Am.sub.2.
[0114] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.2 is Y--R.sub.5
wherein Y is absent and R.sub.5 is maleimido propionic acid.
[0115] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.2 is Y--R.sub.5
wherein Y is --COCH.sub.2(OCH.sub.2CH.sub.2)--NH--, `n` is 2 and
R.sub.5 is maleimido propionic acid.
[0116] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.4 is hydrogen.
[0117] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.4 is
aminoalkyl.
[0118] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.4 is
--NHCOCH.sub.3.
[0119] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which R.sub.4 is
--NHCONH.sub.2.
[0120] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which the peptide bond
(--CONH--) of Ser-Asn is replaced with a modified peptide bond
of
##STR00011##
[0121] wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl group and the alkyl group may be
linear or branched.
[0122] According to yet another embodiment, specifically provided
are compounds of the formula (Ia) in which the peptide bond
(--CONH--) of Asn-Thr is replaced with a modified peptide bond
of
##STR00012##
[0123] wherein Q is hydrogen, --CO(C.sub.1-C.sub.20)alkyl or
--COO(C.sub.1-C.sub.20)alkyl group and the alkyl group may be
linear or branched.
[0124] According to yet another embodiment, specifically provided
are compounds of the formula (Ib) in which R.sub.1 is amidated
C-terminal of Ser.
[0125] According to yet another embodiment, specifically provided
are compounds of the formula (Ib) in which L is
--NH--(CH.sub.2).sub.n--NH-- and `n` is 4.
[0126] According to yet another embodiment, specifically provided
are compounds of the formula (Ib) in which R.sub.4 is hydrogen.
[0127] According to yet another embodiment, specifically provided
are compounds capable of suppressing and/or inhibiting the
programmed cell death 1 (PD1) signalling pathway, wherein the
structures of the compounds are provided in Table 1.
[0128] It should be understood that the formulas (I), (Ia) and (Ib)
structurally encompasses all stereoisomers, enantiomers and
diastereomers, and pharmaceutically acceptable salts that may be
contemplated from the chemical structure of the genera described
herein.
[0129] Compounds of the invention may comprise peptide moieties
that are lipidated and/or are glycosylated. One or more of the
amino acids of the peptide may be a D-amino acid with a view to
increasing stability in vivo.
[0130] The invention includes compounds as described above,
formulated for pharmaceutical administration, typically by
combination with a pharmaceutically acceptable carrier or
diluent.
[0131] The invention includes compounds as described above for use
in a method of medical treatment, e.g. in the treatment of cancer,
treatment of bacterial and viral infections
[0132] The invention further includes a method of screening
compounds for ability to block interaction between PD-1 and a PD-1
ligand, comprising contacting candidate compounds of the kind
described above with PD-1 or a PD-1 ligand binding portion of PD-1
and with a PD-1 ligand or a PD-1 binding portion of a PD-1 ligand,
and measuring the extent of PD-1/PD-1 ligand binding.
[0133] In addition, compounds of the invention may be combined with
carrier molecules such as dendrimers, e.g. PAMAM dendrimers,
liposomes, micro-particles and nanoparticles such as
polycyanoacrylate nanoparticles, and these also may be
PEGylated.
[0134] Below are the representative compounds, which are
illustrative in nature only and are not intended to limit the scope
of the invention.
TABLE-US-00004 TABLE 1 Comp No. Structure 001 ##STR00013## (SEQ ID
NO: 3) 002 ##STR00014## (SEQ ID NO: 4) 003 ##STR00015## (SEQ ID NO:
5) 004 ##STR00016## (SEQ ID NO: 6) 005 ##STR00017## (SEQ ID NO: 7)
006 ##STR00018## (SEQ ID NO: 8) 007 ##STR00019## (SEQ ID NO: 9) 008
##STR00020## (SEQ ID NO: 10) 009 ##STR00021## (SEQ ID NO: 11) 010
##STR00022## *D-Phe (SEQ ID NO: 12) 011 ##STR00023## (SEQ ID NO:
13) 012 ##STR00024## *D-Glu (SEQ ID NO: 14) 013 ##STR00025## *D-Phe
urea (SEQ ID NO: 15) 014 ##STR00026## (SEQ ID NO: 16) 015
##STR00027## *D-Glu (SEQ ID NO: 17) 016 ##STR00028## *All D-amino
acids (SEQ ID NO: 18) 017 ##STR00029## (SEQ ID NO: 19) 018
##STR00030## (SEQ ID NO: 20) 019 ##STR00031## (SEQ ID NO: 21) 020
##STR00032## (SEQ ID NO: 22) 021 ##STR00033## (SEQ ID NO: 23) 022
##STR00034## (SEQ ID NO: 24) 023 ##STR00035## (SEQ ID NO: 25) 024
##STR00036## (SEQ ID NO: 26) 025 ##STR00037## (SEQ ID NO: 27) 026
##STR00038## (SEQ ID NO: 28) 027 ##STR00039## (SEQ ID NO: 29) 028
##STR00040## (SEQ ID NO: 30) 029 ##STR00041## (SEQ ID NO: 31) 030
##STR00042## (SEQ ID NO: 32) 031 ##STR00043## (SEQ ID NO: 33)
[0135] In one of the embodiment of the present invention there is
provided a compound having the ability to inhibit the programmed
cell death 1 (PD1) signalling pathway and being capable of reducing
PD-L1 or PD-L2 binding to PD-1 and resulting immunosuppressive
signalling by PD-1.
[0136] Further embodiment of the present invention relates to the
compounds as disclosed in the present invention, wherein one or
more of the amino acids are substituted with D-amino acid.
[0137] The compounds as disclosed in the present invention are
formulated for pharmaceutical administration.
[0138] Another embodiment of the present invention provided a
pharmaceutical composition comprising the compound as disclosed,
and a pharmaceutically acceptable carrier or diluent.
[0139] Yet another embodiment of the present invention provides use
of the compounds as disclosed in the present invention for the
preparation of a medicament for the treatment of cancer.
[0140] Yet another embodiment of the present invention provides use
of the compounds as disclosed in the present invention for the
preparation of a medicament for the treatment of bacterial, fungal
and viral infection.
[0141] Yet another embodiment of the present invention provides a
method of treatment of cancer, wherein the method comprises
administration of an effective amount of the compound and/or
peptides of the present invention to the subject in need thereof.
Yet another embodiment of the present invention provides a method
for inhibiting growth of tumour cells and/or metastasis by
administering an effective amount of the compound of the present
invention to the subject in need thereof
[0142] The said tumour cells include cancer such as but not limited
to melanoma, renal cancer, prostate cancer, breast cancer, colon
cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer,
cancer of the head or neck, cutaneous or intraocular malignant
melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of
the anal region, stomach cancer, testicular cancer, carcinoma of
the fallopian tubes, carcinoma of the endometrium, carcinoma of the
cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's
Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of
the small intestine, cancer of the endocrine system, cancer of the
thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, sarcoma of soft tissue, cancer of the urethra,
cancer of the penis, chronic or acute leukemias including acute
myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic
leukemia, chronic lymphocytic leukemia, solid tumours of childhood,
lymphocytic lymphoma, cancer of the bladder, cancer of the kidney
or ureter, carcinoma of the renal pelvis, neoplasm of the central
nervous system (CNS), primary CNS lymphoma, tumour angiogenesis,
spinal axis tumour, brain stem glioma, pituitary adenoma, Kaposi's
sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma,
environmentally induced cancers including those induced by
asbestos, and combinations of said cancers.
[0143] Yet another embodiment of the present invention provides a
method of treatment of infection via immunopotentiation caused by
inhibition of immunosuppressive signal induced by PD-1, PD-L1, or
PD-L2, wherein the method comprises administration of an effective
amount of the compound and/or peptides of the present invention to
the subject in need thereof.
[0144] The infectious disease includes but not limited to HIV,
Influenza, Herpes, Giardia, Malaria, Leishmania, the pathogenic
infection by the virus Hepatitis (A, B, & C), herpes virus
(e.g., VZV, HSV-I, HAV-6, HSV-II, CMV and Epstein Barr virus),
adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,
coxsackie virus, cornovirus, respiratory syncytial virus, mumps
virus, rotavirus, measles virus, rubella virus, parvovirus,
vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum
virus, poliovirus, rabies virus, JC virus and arboviral
encephalitis virus, pathogenic infection by the bacteria chlamydia,
rickettsial bacteria, mycobacteria, staphylococci, streptococci,
pneumonococci, meningococci, conococci, klebsiella, proteus,
serratia, pseudomonas, E. coli, legionella, diphtheria, salmonella,
bacilli, cholera, tetanus, botulism, anthrax, plague,
leptospirosis, and Lyme's disease bacteria, pathogenic infection by
the fungi Candida (albicans, krusei, glabrata, tropicalis, etc.),
Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.),
Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii,
Blastomyces dermatitidis, Paracoccidioides brasiliensis,
Coccidioides immitis and Histoplasma capsulatum and pathogenic
infection by the parasites Entamoeba histolytica, Balantidium coli,
Naegleria fowleri, Acanthamoeba sp., Giardia lambia,
Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax,
Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania
donovani, Toxoplasma gondi, Nippostrongylus brasiliensis.
[0145] Still yet another embodiment of the present invention
provides a method for treating sepsis in a subject comprising
administering to the subject a therapeutically effective amount of
compound of the present invention, capable of inhibiting the
programmed cell death 1 (PD1) signaling pathway such that the
subject is treated for the bacterial, fungal and viral
infections.
[0146] The compounds of the present invention may be used as single
drug or as a pharmaceutical composition in which the compound is
mixed with various pharmacologically acceptable materials.
[0147] The pharmaceutical composition is usually administered by a
parenteral administration route, but can be administered by oral or
inhalation routes. Examples of the parenteral administration
include administration by injection, and percutaneous,
transmucosal, transnasal and transpulmonary administrations.
[0148] The injectable materials include a solution, a suspension,
and a solid injection that is dissolved or suspended in a solvent
before use.
[0149] The injection is used after one or more active ingredients
are dissolved, suspended or emulsified in a solvent. Examples of
the solvent include water-soluble solvents (e.g., distilled water,
physiological saline and Ringer's solution), oil solvents (e.g.,
vegetable oils such as olive oil, sesame oil, cotton oil and corn
oil, and alcohols such as propylene glycol, polyethylene glycol and
ethanol), and combinations thereof.
[0150] Further, the injection may contain a stabilizer (e.g., human
serum albumin), solubilizing agent (e.g., polyethylene glycol,
propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol,
trisaminomethane, cholesterol, triethanolamine, sodium carbonate,
sodium citrate, sodium salicylate and sodium acetate), suspending
agent (e.g., surfactants such as stearyl triethanolamine, sodium
lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium
chloride, benzethonium chloride and glyceryl monostearate;
hydrophilic polymers such as polyvinyl alcohol, polyvinyl
pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose,
hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl
cellulose; polysorbates; and polyoxyethylene hardened castor oil),
emulsifier, soothing agent (e.g., benzyl alcohol), tonicity agent
(e.g., sodium chloride, glycerin, D-mannitol, D-sorbitol and
glucose), buffer, preservative (e.g., methylparaben, propylparaben,
benzyl alcohol, chlorobutanol and phenol), antiseptic (e.g.,
paraoxybenzoic esters, chlorobutanol, benzyl alcohol, phenethyl
alcohol, dehydroacetic acid and sorbic acid), antioxidant (e.g.,
sulfite and ascorbate) and dispersant (e.g., Polysorbate 80,
Polyoxyethylene hardened castor oil 60, ethylene glycol,
carboxymethyl cellulose and sodium alginate).
[0151] These injections may be prepared by known methods in the
formulation technology field, such as by a method described in
various Pharmacopoeia. They are prepared, for example, through a
sterilization process at the final stage, or by aseptic
manipulation. It is also possible to use an aseptic solid
formulation, such as a freeze dried product, wherein the aseptic
solid formulation is prepared and dissolved in aseptic or
sterilized distilled water for injection or other solvents before
use.
[0152] These parenteral solutions may be supplied in a vessel with
a standard capacity, such as a plastic or glass vial, ampoule,
syringe and injector, or in a vessel with a large capacity, such as
a bottle.
[0153] The dosage of the compounds of the present invention varies
depending on age, weight, symptom, therapeutic efficacy, dosing
regimen and/or treatment time. Generally, they may be administered
by a parenteral route (preferably intravenous administration) in an
amount of 1 mg to 100 mg per time, from once a couple of days, once
3 days, once 2 days, once a day to a couple of times a day, in the
case of an adult, or continuously administered by intravenous
administration from 1 to 24 hours a day. Since the dosage is
affected by various conditions, an amount less than the above
dosage may sometimes work well enough, or higher dosage may be
required in some cases.
[0154] Parenteral administration by injection includes all forms of
injections, and also includes intravenous fluids. For example, it
includes intramuscular injections, subcutaneous injections,
intradermal injections, intraarterial injections, intravenous
injections, intraperitoneal injections, injections to spinal
cavity, and intravenous drops.
[0155] The compounds of the present invention may be administered
in combination with other drugs for (1) complementation and/or
enhancement of prevention and/or therapeutic efficacy of the
preventive and/or therapeutic drug of the present invention, (2)
dynamics, absorption improvement, dosage reduction of the
preventive and/or therapeutic drug of the present invention, and/or
(3) reduction of the side effects of the preventive and/or
therapeutic drug of the present invention.
[0156] A concomitant medicine comprising the peptide of the present
invention and other drug may be administered as a combination
preparation in which both components are contained in a single
formulation, or administered as separate formulations. The
administration by separate formulations includes simultaneous
administration and administration with some time intervals. In the
case of the administration with some time intervals, the compound
of the present invention can be administered first, followed by
another drug or another drug can be administered first, followed by
the compound of the present invention. The administration method of
the respective drugs may be the same or different.
[0157] The dosage of the other drug can be properly selected, based
on a dosage that has been clinically used. The compounding ratio of
the compound of the present invention and the other drug can be
properly selected according to age and weight of a subject to be
administered, administration method, administration time, disorder
to be treated, symptom and combination thereof. For example, the
other drug may be used in an amount of 0.01 to 100 parts by mass,
based on 1 part by mass of the compound of the present invention.
The other drug may be a combination of two or more kind of
arbitrary drugs in a proper proportion. The other drug that
complements and/or enhances the preventive and/or therapeutic
efficacy of the compound of the present invention includes not only
those that have already been discovered, but those that will be
discovered in future, based on the above mechanism.
[0158] Diseases on which this concomitant use exerts a preventive
and/or therapeutic effect are not particularly limited. The
concomitant medicine can be used for any diseases, as long as it
complements and/or enhances the preventive and/or therapeutic
efficacy of the compound of the present invention.
[0159] Particularly, since the compound of the present invention
exhibits an effect of stimulating or proliferating lymphoid cells,
the concomitant use is able to reduce a dosage of chemotherapeutics
commonly used or an irradiation dosage in radio therapy. This
results in suppression of side effects that accompany with
chemotherapy and radio therapy.
[0160] The compound of the present invention can be used with an
existing chemotherapeutic concomitantly or in a mixture form.
Examples of the chemotherapeutic include an alkylation agent,
nitrosourea agent, antimetabolite, anticancer antibiotics,
vegetable-origin alkaloid, topoisomerase inhibitor, hormone drug,
hormone antagonist, aromatase inhibitor, P-glycoprotein inhibitor,
platinum complex derivative, other immunotherapeutic drugs and
other anticancer drugs. Further, it can be used with a cancer
treatment adjunct, such as a leucopenia (neutrophenia) treatment
drug, thrombocytopenia treatment drug, antiemetic and cancer pain
intervention drug, concomitantly or in a mixture form.
[0161] The compound of the present invention can be used with other
immunomodulators concomitantly or in a mixture form. Examples of
the immunomodulator include various cytokines Examples of the
cytokines that stimulates immune responses include GM-CSF, M-CSF,
G-CSF, interferon-.alpha., .beta., or .gamma., IL-1, IL-2, IL-3 and
IL-12.
[0162] The concomitant use of the compound of the present invention
and a cancer antigen is able to give an additive or synergetic
enhancement effect. Examples of the cancer antigen include HLA-A1
and HLA-A2 derived peptides derived from MAGE-1 or
[0163] MAGE-3 of malignant melanoma, MART-1 and gp100, HER2/neu
peptide of breast cancer and ovarian cancer, MUC-1 peptide of
adenocarcinoma and NY-ESO--1 of metastatic cancer.
EXPERIMENTAL
Purification and Characterization of Peptide
[0164] The Reverse phase analytical HPLC was performed using on
Zorbax Eclipse XDB-C18 silica column (4.6 mm.times.250 mm, 5
.mu.m). Buffer A: 0.1% TFA/Water, Buffer B: 0.1% TFA in 9:1
acetonitrile/water. Equilibration of the column with 2% buffer B
and elution by a gradient of 2% to 25% buffer B in 5 min and from
25% to 40% buffer B in total run time of 20 min.
[0165] LCMS was performed on AP1 2000 LC/MS/MS triple quad (Applied
biosystems) with Agilent 1100 series HPLC with G1315 B DAD diode
array detector, using Mercury MS column.
[0166] For further illustration of methods of preparing the
compounds of the present invention, the following examples are
disclosed below.
Example 1
Synthesis of Compound 1
##STR00044##
[0168] Resin:
[0169] 1 g CLEAR Amide Resin (RCY 1250-PI, Lot No. 224481), 0.46
mmol/g
[0170] Desiccated CLEAR Amide resin (0.46 mmol/g, 1 g) was placed
in a polyethylene vessel equipped with a polypropylene filter.
Resin was swelled in DCM (15 mL) for 1 h and DMF (15 mL) for 1 h.
The Fmoc group of the CLEAR Amide was deprotected by treating it
twice with 20% (v/v) piperdine/DMF solution for 5 and 15 min (10
mL). The resin was washed with DMF (6.times.10 mL), DCM (6.times.10
mL) and DMF (6.times.10 mL). Kaiser test on peptide resin aliquot
upon completion of Fmoc-deprotection was positive. The C-terminal
amino acid, Fmoc-Glu(OAllyl)-OH (0.98 g; 5 equiv. 2.3 mmol) in dry
DMF was added to the deprotected resin and coupling was initiated
with DIC (0.36 mL; 5 equiv) and HOBT (0.32 g; 5 equiv) in DMF. The
concentration of each reactant in the reaction mixture was
approximately 0.4 M. The mixture was rotated on a rotor at room
temperature for 2 h. Resin was filtered and washed with DMF
(6.times.10 mL), DCM (6.times.10 mL) and DMF (6.times.10 mL).
Kaiser test on peptide resin aliquot upon completion of coupling
was negative. After the first amino acid attachment, the un-reacted
amino group, if any, in the resin is capped, used acetic
anhydride/pyridine/DCM (1:8:8) for 20 minutes to avoid any deletion
of the sequence. After capping, resin was washed with DCM
(6.times.10 mL), DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF
(6.times.10 mL). The Fmoc group on the C-terminal amino acid
attached peptidyl resin was deprotected by treating it twice with
20% (v/v) piperdine/DMF solution for 5 and 15 min (10 mL). The
resin was washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and
DMF (6.times.10 m L). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. Fmoc-Ser (OtBu)-OH
(0.9 g; 5 equiv. 2.3 m mol) in dry DMF was added to the deprotected
resin and coupling was initiated with DIC (0.36 mL; 5 equiv) and
HOBT (0.32 g; 5 equiv) in DMF. The concentration of each reactant
in the reaction mixture was approximately 0.4 M. The mixture was
rotated on a rotor at room temperature for 2 h. Resin was filtered
and washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF
(6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of coupling was negative. The Fmoc group of the peptidyl
resin was deprotected by treating it twice with 20% (v/v)
piperdine/DMF solution for 5 and 15 min (10 mL). The resin was
washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF
(6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. Next amino acid in
the peptide sequence Fmoc-Thr (OtBu)-OH (0.92 g; 5 equiv. 2.3 m
mol) in dry DMF was added to the deprotected resin and coupling was
initiated with DIC (0.36 mL; 5 equiv) and HOBT (0.32 g; 5 equiv) in
DMF. The concentration of each reactant in the reaction mixture was
approximately 0.4 M. The mixture was rotated on a rotor at room
temperature for 2 h. Resin was filtered and washed with DMF
(6.times.10 m L), DCM (6.times.10 mL) and DMF (6.times.10 mL).
Kaiser test on peptide resin aliquot upon completion of coupling
was negative. On completion of threonine coupling Fmoc group on the
threonine was deprotected by treating it twice with 20% (v/v)
piperdine/DMF solution for 5 and 15 min (10 mL). The resin was
washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF
(6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. Next amino acid
Fmoc-Asn (Trt)-OH (1.4 g; 5 equiv. 2.3 mmol) in dry DMF was added
to the deprotected resin and coupling was initiated with DIC (0.36
mL; 5 equiv) and HOBT (0.32 g; 5 equiv) in DMF. The concentration
of each reactant in the reaction mixture was approximately 0.4 M.
The mixture was rotated on a rotor at room temperature for 2 h.
Resin was filtered and washed with DMF (6.times.10 mL), DCM
(6.times.10 mL) and DMF (6.times.10 mL). Kaiser test on peptide
resin aliquot upon completion of coupling was negative. The Fmoc
group on the peptidyl resin was deprotected by treating it twice
with 20% (v/v) piperdine/DMF solution for 5 and 15 min (10 mL). The
resin was washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and
DMF (6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. Boc-Ser (OtBu)-OH
(0.62 g; 5 equiv. 2.3 m mol) in Dry DMF was added to the
deprotected resin and coupling was initiated with DIC (0.36 mL; 5
equiv) and HOBT (0.32 g; 5 equiv) in DMF. The concentration of each
reactant in the reaction mixture was approximately 0.4 M. The
mixture was rotated on a rotor at room temperature for 2 h. Resin
was filtered and washed with DMF (6.times.10 mL), DCM (6.times.10
mL) and DMF (6.times.10 mL). Kaiser test on peptide resin aliquot
upon completion of coupling was negative. The linear chain of the
sequence is completed and further chain elongation is achieved by
removing the orthogonal ally ester protecting group on the Glutamic
acid.
[0171] Removal of Allyl Ester (OAll) Protecting Group on the Side
Chain Carboxyl Acid of Glutamic Acid
[0172] After the completion of the linear protected peptide
sequence, the Allyl protecting group from the carboxyl moiety of
Glu was removed from the peptidyl resin by treating with
tetrakistriphenylphosphine palladium (0) (10 Equiv; 5.3 g) and
Phenylsilane (20 eqv; 1.2 m L) in a solution of
chloroform/N-methylpyrrolidine (95/5 v/v) for 4 h under argon. The
resin was washed with a solution of 10% NMP in chloroform
(6.times.10 mL), 1% DIEPA in DMF (6.times.10 mL), DCM (6.times.10
mL), DMF (6.times.10 mL).
[0173] Attachment of Linker Fmoc-1,4-Diamino Butane Hydrochloride
to the Carboxyl End of Glutamic Acid
[0174] Fmoc 1,4-diamino butane HCL (0.24 g; 1.5 equiv. 0.69 mmol)
in Dry DMF was added to the deprotected resin and coupling was
initiated with DIC (0.18 mL; 1.2 m mol equiv) and HOBT (0.08 g;
0.69 m mol) in DMF. The concentration of each reactant in the
reaction mixture was approximately 0.4 M. The mixture was rotated
on a rotor at room temperature for overnight. Resin was filtered
and washed with DMF (6.times.10 mL), DCM (6.times.15 mL) and DMF
(6.times.10 mL). The Fmoc group of the linker attached to peptidyl
resin was deprotected by treating it twice with 20% (v/v)
piperdine/DMF solution for 5 and 15 min (10 mL). The resin was
washed with DMF (6.times.10 mL), DCM (6.times.10 m L) and DMF
(6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. Next amino acid
Fmoc-Phe-OH (0.89 g; 5 equiv. 2.3 mmol) in dry DMF was added to the
deprotected resin and coupling was initiated with DIC (0.43 mL; 6
equiv) and HOBT (0.32 g; 5 equiv) in DMF. The concentration of each
reactant in the reaction mixture was approximately 0.4 M. The
mixture was rotated on a rotor at room temperature for 2 h. Resin
was filtered and washed with DMF (6.times.10 m L), DCM (6.times.10
mL) and DMF (6.times.10 mL). Kaiser test on peptide resin aliquot
upon completion of coupling was negative. The Fmoc group of the
peptidyl resin was deprotected by treating it twice with 20% (v/v)
piperdine/DMF solution for 5 and 15 min (10 mL). The resin was
washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF
(6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. The last amino acid
of the complete sequence Boc-Ser (OtBu)-OH (0.62 g; 5 equiv. 2.3
mmol) in dry DMF was added to the deprotected resin and coupling
was initiated with DIC (0.43 mL; 6 equiv) and HOBT (0.32 g; 5
equiv) in DMF. The concentration of each reactant in the reaction
mixture was approximately 0.4 M. The mixture was rotated on a rotor
at room temperature for 2 h. Resin was filtered and washed with DMF
DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF (6.times.10 mL).
Kaiser test on peptide resin aliquot upon completion of coupling
was negative.
[0175] Final Cleavage of Peptide from the Resin
[0176] The peptidyl resin was washed with, DCM (6.times.10 mL),
MeOH (6.times.10 mL) and ether (6.times.10 mL) and dried in vacuum
desiccators overnight. The cleavage of the peptide from the solid
support was achieved by treating the peptide-resin with reagent
cocktail (90.0% TFA/5% TIPS/5% H.sub.2O) at room temperature for
2.5 h. Cleavage mixture was collected by filtration and the resin
was washed with TFA (2 mL) and DCM (2.times.5 mL).
[0177] The excess TFA and DCM was concentrated to small volume
under nitrogen and a small amount of DCM (5-10 mL) was added to the
residue and evaporated under nitrogen. The process was repeated 3-4
times to remove most of the volatile impurities. The residue was
cooled to 0.degree. C. and anhydrous ether was added to precipitate
the peptide. The precipitated peptide was centrifuged and the
supernatant ether was removed and fresh ether was added to the
peptide and re-centrifuged (270 mg, 70% yield). The residue was
dissolved in Millipore water and lyophilized to obtain the crude
peptide. The crude sample was preparative HPLC purified on Zorbax
Eclipse XDB-C18 column (9.4 mm.times.250 mm, 5 .mu.m) with buffer
A: 0.1% TFA/Water, buffer B: Acetonitrile. The peptide was eluted
by gradient elution 0-5 min=5% buffer B, 5-25 min=5-60% buffer B
with a flow rate of 7 mL/min. The identity of peptide was confirmed
by LCMS. Calculated Mass: 840, Observed Mass: 840.4[M].sup.+.
Example 2
Synthesis of Compound 2
##STR00045##
[0179] The synthesis was carried out manually using the same method
as in example 1 using desiccated Rink Amide MBHA-Amide resin
(100-200 mesh, 0.66 mmol/g, 0.75 g). The N-terminal amino acid
serine was coupled as Fmoc-Ser-(OtBu)-OH as detailed in example 1.
The Fmoc group of the linker attached to peptidyl resin was
deprotected by treating it twice with 20% (v/v) piperdine/DMF
solution for 5 and 15 min (10 mL). The resin was washed with DMF
(6.times.10 mL), DCM (6.times.10 mL) and DMF (6.times.10 mL).
Kaiser test on peptide resin aliquot upon completion of
Fmoc-deprotection was positive. The free amine was acetylated using
acetic anhydride/pyridine/DCM (1:8:8) for 20 minutes. After
acetylation, resin is washed with DCM (6.times.10 mL); DMF
(6.times.10 mL), DCM (6.times.10 mL), and DMF (6.times.10 mL).
Kaiser test on peptide resin aliquot upon completion of coupling
was negative. The linear chain of the sequence is completed and
further chain elongation is achieved by removing the orthogonal
allyl ester protecting group on the Glutamic acid as mentioned in
example 1 to yield 300 mg, 70% crude peptide (300 mg, 70% yield).
The crude sample was preparative HPLC purified on Zorbax Eclipse
XDB-C18 column (9.4 mm.times.250 mm, 5 .mu.m) with buffer A: 0.1%
TFA/Water, buffer B: Acetonitrile. The peptide was eluted by
gradient elution 0-8 min=5-15% buffer B, 8-10 min=15-20% buffer B
with a flow rate of 7 mL/min The identity of peptide was confirmed
by LCMS. Calculated Mass: 882.3, Observed Mass: 882.6
[M].sup.+.
[0180] Synthesis of Building Block
##STR00046##
[0181] To a solution of L-Serine (1.25 g, 11.8 mmol) in 8.5 mL of
TFA, Hydroquonone (catalytic-100 mg) was added at 0.degree. C. The
resulting mixture was stirred at room temperature for 10 min, to
this solution at room temperature decanoyl chloride (3.4 g, 17.8
mmol) was added slowly and the reaction was allowed to continue for
2 h. After the completion of 2 h, the reaction mixture was diluted
with diethylether (100 mL) and triturated at 0-50.degree. C. to get
white precipitate, which was centrifuged and dried under vacuum to
furnish desired compound as white solid [yield: 3.1 g, 88.6%;
ELSD-HPLC: 99%, Mass: Cal. 259.18, Obs--260.2]
[0182] Acid-amine hydrochloride A (2.5 g, 8.4 mmol) was dissolved
in DCM (25 mL) together with catalytic amount of hydroquinone (10
mg), and poured in to a solution of di-tert-butyl dicarbonate (1.84
g, 8.4 mmol) and triethyl amine (3.5 mL, 25.4 mmol) in DCM (20 mL).
The solution was then refluxed for 30 min to give a clear solution
and cooled in an ice bath. A solution of NaHSO.sub.4 (4 g in 40 mL
water) was added, the reaction mixture was then partitioned between
water and DCM, and the aqueous phase was extracted once again with
DCM (25 mL). The combined organic phases were washed with brine,
dried over Na.sub.2SO.sub.4 and evaporated in vacuo to give desired
compound as colourless oil. (Yield: 3.0 g, 98.0%; ELSD-HPLC: 99%,
Mass: Cal. 359.2, Obs--382 (M+Na)].
Example 3
Synthesis of Compound 4
##STR00047##
[0184] The synthesis was carried out as explained in example 1
using Rink Amide MBHA Resin (RFR-1063-PI Lot No 2401691), 0.66
mmol/g, 0.75 g. After the completion of linear synthesis,
orthogonal deprotection, coupling of phenyl alanine and serine was
carried out as in example 1. The N-terminus serine in the branch
was coupled as Fmoc-Ser(OtBu)-OH and on completion of serine
coupling as evidenced by Kaiser test, Fmoc-deprotection was carried
out. The next residue 3-malemidopropanoic acid (0.17 g; 2 equiv.
0.99 mmol) in dry DMF was added to the deprotected resin and
coupling was initiated with DIC (0.23 mL; 3 equiv) and HOBT (0.13
g; 2 equiv) in DMF. The concentration of each reactant in the
reaction mixture was approximately 0.4 M. The mixture was rotated
on a rotor at room temperature for overnight. Resin was filtered
and washed with DMF DMF (6.times.10 mL), DCM (6.times.10 mL) and
DMF (6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of coupling was negative. The final cleavage of peptidyl
resin was carried out as in example 1 to yield 300 mg, 70% yield of
crude peptide. The crude sample was preparative HPLC purified on
Zorbax Eclipse XDB-C18 column (9.4 mm.times.250 mm, 5 .mu.m) with
buffer A: 0.1% TFA/Water, buffer B: Acetonitrile. The peptide was
eluted by gradient elution 0-5 min=5-10% buffer B, 5-9 min=10-25%
buffer B with a flow rate of 7 mL/min. The identity of peptide was
confirmed by LCMS. Calculated Mass: 990.3, Observed Mass:
991.4[M+H].sup.+.
Example 4
Synthesis of Compound 16
##STR00048##
[0186] The synthesis was carried out as explained in example 1
using Rink Amide MBHA Resin (RFR-1063-PI Lot No 2401691), 0.66
mmol/g, and 0.75 g. In this example Fmoc-D-Ser(OtBu) was coupled as
the C-terminal amino acid. The synthesis was continued using
D-amino acids. D-Boc-Glu(OAllyl)-OH was used as the building block
to incorporate Glutamic acid. Coupling of amino acids and cleavage
of peptidyl resin was carried out as mentioned in example 1 to
yield 300 mg, 70% yield of crude peptide The crude sample was
preparative HPLC purified on Zorbax Eclipse XDB-C18 column (9.4
mm.times.250 mm, 5 .mu.m) with buffer A: 0.1% TFA/Water, buffer B:
Acetonitrile. The peptide was eluted by gradient elution 0-9
min=5-20% buffer B, with a flow rate of 7 mL/min. The identity of
peptide was confirmed by LCMS. Calculated Mass: 840, Observed Mass:
841[M+H].sup.+.
[0187] Synthesis of Building Block
##STR00049##
[0188] Compound E (1.0 g, 1.44 mmol) was dissolved in 20 mL of DCM
containing triethyl amine (0.218 g, 2.16 mmol) at -20.degree. C. At
same temperature, isobutyl chloroformate (0.216 g, 1.5 mmol) was
added slowly, and the mixture was stirred for 1 h at -20.degree. C.
under N.sub.2 atmosphere. A further quantity of isobutyl
chloroformate (0.216 g, 1.5 mmol) and triethyl amine (0.218 g, 2.16
mmol) was added and stirring was continued at 0.degree. C. until
the reactants were consumed as determined by TLC analysis. The
reaction mixture concentrated under reduced pressure and diluted
with EtOAc, the organic extract was washed with water (50 mL x 2),
brine (50 mL) and dried (Na.sub.2SO.sub.4). The solvent was
evaporated to give the crude product which was further purified by
silica gel column chromatography (eluent: 0-40% EtOAc in Hexane) to
furnish the product I (yield: 0.92 g,: 80.7%; Mass: Cal. 793.43,
Obs--794.3 (M+1), 817.3 (M+Na).
##STR00050##
[0189] To a solution of compound I (0.68 g) in methanol (10 mL)
under inert atmosphere was added 10% Pd--C (0.25 g), and the
mixture was stirred for 4 h under H, atmosphere. The completion of
the reaction was confirmed by TLC analysis. After the completion of
reaction, the catalyst was removed by filtration through a celite
pad, which was then washed with 30 mL of methanol. The combined
organic filtrate, on evaporation under reduced pressure resulted in
the isolation of pure product J (yield: 0.58 g, 96%, Mass: Cal.
703.38, Obs--704.3 (M+1), 726.3 (M+Na).
Example 5
Synthesis of Compound 18
##STR00051##
[0191] The synthesis was carried out as explained in example 1
using Rink Amide MBHA Resin (RFR-1063-PI Lot No 2401691), 0.66
mmol/g, 0.75 g. The N-terminal amino acid in the linear chain was
coupled as Boc-Ser-(OCO(CH.sub.2).sub.8CH.sub.3)--OH (compound B,
0.27 g; 1.5 equiv. 0.745 mmol) in dry DMF was added to the
deprotected resin and coupling was initiated with DIC (0.2 mL; 2.5
equiv) and HOBT (0.1 g; 1.5 equiv) in DMF. The concentration of
each reactant in the reaction mixture was approximately 0.4 M. The
mixture was rotated on a rotor at room temperature for overnight.
Resin was filtered and washed with DMF (6.times.10 mL), DCM
(6.times.10 mL) and DMF (6.times.10 mL). After the completion of
the linear protected peptide sequence, the Allyl protecting group
from carboxyl moiety of Glutamic acid was removed from the peptidyl
resin and synthesis continued in the branch as mentioned in example
1 to yield 363 mg, 75% yield of crude peptide. The crude sample was
preparative HPLC purified on Zorbax Eclipse XDB-C18 column (9.4
mm.times.250 mm, 5 .mu.m) with buffer A: 0.1% TFA/Water, buffer B:
Acetonitrile. The peptide was eluted by gradient elution 0-5
min=5-15% buffer B, 5-10 min=15-25% buffer B with a flow rate of 7
mL/min. The identity of peptide was confirmed by LCMS. Calculated
Mass: 980, observed mass 980.4 [M].sup.+
[0192] Synthesis of Building Block
##STR00052##
[0193] To a solution of N-Boc amino acid (10.0 g, 38.28 mmol) in
THF (100 mL) at -20.degree. C., was added N-methyl morpholine (NMM,
4.25 g, 42.11 mmol) and ethylchloroformate (4.57 g, 42.11 mmol) and
the resultant mixture was stirred at same temperature for 20 min.
The inorganic salts were filtered off and the filtrate was treated
with moist NaBH.sub.4 (2.9 g, 76.56 mmol) for 10-15 min. The
reaction mixture was then partitioned between water and EtOAc.
Organic layer was washed with water, 10% NaHCO.sub.3 solution (100
mL x 2) and brine, dried over Na.sub.2SO.sub.4 and evaporated under
reduced pressure to yield N-Boc aminol, which was further purified
by silica gel column chromatography (eluent: 0-50% EtOAc in Hexane)
to yield 8.2 g of product [yield: 85.4%, Mass: Cal. 247.3, Obs:
248.2 (M+1), 270.2 (M+Na)].
##STR00053##
[0194] To the solution of N-Boc-aminol (3.0 g, 12.13 mmol) in
distilled DCM (30.0 mL) was added Dess-Martin periodinane (10.3 g,
24.27 mmol) in a portion wise manner at 0.degree. C. and stirred at
rt under N.sub.2 atmosphere for 30 min until the reactants were
consumed as determined by TLC analysis. The reaction mixture was
quenched by adding 1.0M Na.sub.2S.sub.2O.sub.3 solution, and the
product was extracted with DCM. The organic extract was washed with
(5%, 1:1) Na.sub.2S.sub.2O.sub.3/NaHCO.sub.3 solution (20 mL x 2),
brine (20 mL) and dried over Na.sub.2SO.sub.4. The solvent was
removed under reduced pressure to give the crude product, which was
further purified by silica gel column chromatography (eluent: 0-20%
EtOAc in Hexane) to yield 2.4 g pure product C (yield-82%,
Mass-Cal. 245.32, Obs--247.9 (M+1), 265.1 (M+Na)).
##STR00054##
[0195] To a solution of Fmoc-Asn(trt)-OH (4.0 g, 6.7 mmol) in 30.0
mL of DMF was added Cs.sub.2CO.sub.3 (2.62 g, 8.0 mmol). The
mixture was then cooled to 0.degree. C. and benzyl bromide (1.37 g,
8.0 mmol) was added and the resultant solution was stirred for 30
min at 0.degree. C. and then at rt for 12 h. The reaction mixture
was concentrated under reduced pressure and diluted with EtOAc (50
mL), the organic layer was washed with NaHCO.sub.3 (2.times.50 m L)
and brine (1.times.50 mL), dried over Na.sub.2SO.sub.4, filtered
and concentrated in vacuo and purified by silica gel column
chromatography (eluent: 0-30% EtOAc in Hexane) to furnish
Fmoc-Asn(trt)-OBn as white solid [yield: 4.5 g, 98.0%; Mass: Cal.
686.28, Obs--687.3 (M+1), 709.1 (M+Na].
[0196] To a solution of Fmoc-Asn(Trt)-OBn (3.5 g, 5.1 mmol) in DCM
(14.0 mL), diethylamine (14.0 mL) was added and stirred for 1 h at
rt. The resulting solution was concentrated in vacuo and the
thick-residue was purified by neutral alumina column chromatography
(eluent: 0-50% EtOAc in Hexane then 0-5% MeOH in CHCl.sub.3) to
yield NH.sub.2-Asn(Trt)-OBn D (yield: 1.75 g, 73.0%; Mass: Cal.
464.21, Obs--465.3 (M+1), 487.2 (M+Na]).
##STR00055##
[0197] Asn(Trt)-OBn (4.5 g, 9.7 mmol), DIPEA (2.5 g, 19.4 mmol) and
Boc-Ser(OtBu)-CHO (2.4 g, 9.7 mmol) were mixed in DCM (45 mL) at
0.degree. C. and then allowed to stir at room temperature for 1 h.
Again the reaction mixture was cooled to 0.degree. C. and treated
with sodium triacetoxyborohydride (4.1 g, 19.4 mmol) and then
mixture was allowed to stir at room temperature under N.sub.2
atmosphere for 6 h until the reactants were consumed as determined
by TLC analysis. The reaction mixture was quenched by adding water,
and the product was extracted with DCM. The organic extract was
washed with 5% NaHCO.sub.3 solution (50 mL x 2), 5% citric acid
solution (50 mL x 2), brine (50 mL) and dried over
Na.sub.2SO.sub.4. The solvent was evaporated to give the crude
product which was further purified by silica gel column
chromatography (eluent: 5-40% EtOAc in Hexane) to furnish the
desired product E (yield: 4.2 g, 62.0%; Mass: Cal. 693.27,
Obs--694.4 (M+1), 716.0 (M+Na)).
##STR00056##
[0198] To a solution of compound E (4.0 g) in methanol (70.0 mL)
under inert atmosphere, was added 10% Pd--C (1.0 g) and the mixture
was stirred for 4 h under H.sub.2 atmosphere. The completion of the
reaction was confirmed by TLC analysis. The catalyst was then
removed by filtration through a celite pad, which was then washed
with 50 mL of methanol. The combined organic filtrate, on
evaporation under reduced pressure resulted in the isolation of the
product F, (Yield: 3.3 g, 96.0%; Mass: Cal. 603.25, Obs--604.4
(M+1), 626.4 (M+Na).
##STR00057##
[0199] Compound E (0.96 g, 1.38 mmol) was dissolved in 20 mL of DCM
containing triethyl amine (0.384 g, 2.7 mmol) at 0.degree. C. At
same temperature, decanoyl chloride (0.263 g, 1.38 mmol) was added
slowly, and the mixture was stirred for 5 min at 0.degree. C., and
at rt for 1 h under N.sub.2 atmosphere. A further quantity of
decanoyl chloride (0.263 g, 1.38 mmol) and triethyl amine (0.384 g,
2.7 mmol) was added and stirring was continued until the reactants
were consumed as determined by TLC analysis. The reaction mixture
was quenched by adding water, and the product was extracted with
DCM. The organic extract was washed with water (50 mL x 2), brine
(50 mL) and dried (Na.sub.2SO.sub.4). The solvent was evaporated to
give the crude product which was further purified by silica gel
column chromatography (eluent: 0-40% EtOAc in Hexane) to furnish
the product G (yield: 0.87 g, 81.9%; Mass: Cal. 847.2, Obs--848.3
(M+1), 870.4 (M+Na).
##STR00058##
[0200] To a solution of compound G (0.72 g, 0.85 mmol) in methanol
(10 mL) under inert atmosphere was added 10% Pd--C (0.25 g), and
the mixture was stirred for 4 h under H.sub.2 atmosphere. The
completion of the reaction was confirmed by TLC analysis. After the
completion of reaction, the catalyst was removed by filtration
through a celite pad, which was then washed with 30 mL of methanol.
The combined organic filtrate, on evaporation under reduced
pressure resulted in the isolation of pure product H (yield: 0.58
g, 90.6%, Mass: Cal. 757.3, Obs--758.2 (M+1), 780.3 (M+Na).
Example 6
Synthesis of Compound 20
##STR00059##
[0202] The synthesis was carried out as explained in example 1
using Rink Amide MBHA Resin (RFR-1063-PI Lot No 2401691), 0.66
mmol/g, 0.75 g. The N-terminal amino acid in the linear chain was
coupled as (compound H, 0.56 g; 1.5 equiv. 0.74 m mol) in Dry DMF
was added to the deprotected resin and coupling was initiated with
DIC (0.2 mL; 2.5 equiv) and HOBT (0.1 g; 1.5 equiv) in DMF. The
concentration of each reactant in the reaction mixture was
approximately 0.4 M. The mixture was rotated on a rotor at room
temperature for overnight. Resin was filtered and washed with DMF
(6.times.10 mL), DCM (6.times.10 mL) and DMF (6.times.10 mL). After
the completion of the linear protected peptide sequence, the
orthogonal deprotection, attachment of linker and coupling of
phenyl alanine and serine was carried out as in example 1. The
peptide was cleaved from the polymeric support to yield 300 mg, 66%
yield of crude peptide. The crude sample was preparative HPLC
purified on Zorbax Eclipse XDB-C18 column (9.4 mm.times.250 mm, 5
.mu.m) with buffer A: 0.1% TFA/Water, buffer B: Acetonitrile. The
peptide was eluted by gradient elution 0-5 min=5-15% buffer B, 5-10
min=15-25% buffer B with a flow rate of 7 mL/min. The identity of
peptide was confirmed by LCMS. Calculated Mass: 925.5 Observed
Mass: 926.7 [M+H].sup.+
Example 7
Synthesis of Compound 26
##STR00060##
[0204] The synthesis was carried out as explained in example 1
using Rink Amide MBHA Resin (RFR-1063-PI Lot No 2401691), 0.66
mmol/g, and 0.75 g. The N-terminal amino acid in the linear
fragment was coupled as (compound J, 0.52 g; 1.5 equiv. 0.745 mmol)
in dry DMF was added to the deprotected resin and coupling was
initiated with DIC (0.2 m L; 2.5 equiv) and HOBT (0.1 g; 1.5 equiv)
in DMF. The concentration of each reactant in the reaction mixture
was approximately 0.4 M. The mixture was rotated on a rotor at room
temperature for overnight. Resin was filtered and washed with DMF
(6.times.10 mL), DCM (6.times.10 mL) and DMF (6.times.10 mL). The
synthesis was continued further after OAR deprotection and coupling
of branch amino acids as explained in the example 1 to yield 363
mg, 75% yield of crude peptide. The crude sample was preparative
HPLC purified on Zorbax Eclipse XDB-C18 column (9.4 mm.times.250
mm, 5 .mu.m) with buffer A: 0.1% TFA/Water, buffer B: Acetonitrile.
The peptide was eluted by gradient elution 0-5 min=5-10% buffer B,
5-20 min=10-25% buffer B with a flow rate of 7 mL/min. The identity
of peptide was confirmed by LCMS. Calculated Mass: 925.5, observed
mass 926.7 [M+H].sup.+.
Example 8
Synthesis of Compound 28
##STR00061##
[0206] Desiccated Rink Amide MBHA-Amide resin (100-200 mesh, 0.66
mmol/g, 0.5 g) was placed in a polyethylene vessel equipped with a
polypropylene filter. Resin was swelled in DCM (15 mL) for 1 h and
DMF (15 mL) for 1 h. The Fmoc group of the Rink Amide
[0207] MBHA-Amide was deprotected by treating it twice with 20%
(v/v) piperidine/DMF solution for 5 and 15 min (10 mL). The resin
was washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and DMF
(6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deportation was positive. The C-terminal amino
acid, Fmoc-Ser(tBu)-OH (0.64 g; 5 equiv. 1.65 mmol) in dry DMF was
added to the deprotected resin and coupling was initiated with DIC
(0.26 mL; 5 equiv) and HOBT (0.23 g; 5 equiv) in DMF. The
concentration of each reactant in the reaction mixture was
approximately 0.4 M. The mixture was rotated on a rotor at room
temperature for 2 h. Resin was filtered and washed with DMF
(6.times.10 mL), DCM (6.times.10 mL) and DMF (6.times.10 mL).
Kaiser test on peptide resin aliquot upon completion of coupling
was negative After the first amino acid attachment, the unreacted
amino group, if any, in the resin is capped used acetic
anhydride/pyridine/DCM (1:8:8) for 20 minutes to avoid any deletion
of the sequence. After capping, resin is washed with DCM
(6.times.10 mL), DMF (6.times.10 mL), DCM (6.times.15 mL) and DMF
(6.times.15 mL). The Fmoc group on the C-terminal amino acid
attached peptidyl resin was deprotected by treating it twice with
20% (v/v) piperdine/DMF solution for 5 and 15 min (15 mL). The
resin was washed with DMF (6.times.10 mL), DCM (6.times.10 mL) and
DMF (6.times.10 mL). Kaiser test on peptide resin aliquot upon
completion of Fmoc-deprotection was positive. The remaining amino
acids were attached to the solid support as mentioned in example 1.
The following amino acids were added sequentially to the peptidyl
resin; Fmoc-Phe-OH (0.64 g; 5 equiv. 1.65 mmol), Boc-Lys(Fmoc)-OH
(0.78 g; 5 equiv. 1.65 mmol), Fmoc-Glu(OtBu)-OH (0.7 g; 5 equiv.
1.65 mmol), Fmoc-Ser(tBu)-OH (0.64 g; 5 equiv. 1.65 mmol),
Fmoc-Thr(OtBu)-OH (0.66 g; 5 equiv. 1.65 mmol), Fmoc-Asn(Trt)-OH
(0.98 g; 5 equiv. 1.65 mmol), Fmoc-Ser (OtBu)-OH (0.64 g; 5 equiv.
1.65 mmol). The coupling was carried out in dry DMF using DIC (0.26
mL; 5 equiv) and HOBT (0.23 g; 5 equiv) in DMF. The cleavage of the
peptidyl resin was carried out as mentioned in example 1. The
precipitated peptide was centrifuged and the supernatant ether was
removed and fresh ether was added to the peptide and
re-centrifuged. The residue was dissolved in Millipore water and
lyophilized to obtain the crude peptide (222 mg, 75% yield). The
crude sample was preparative HPLC purified and Lyophilised. The
identity of peptide was confirmed by LCMS. Calculated Mass: 897.4,
Observed Mass: 898.4 [M+1].sup.+.
[0208] The following compounds were prepared by following similar
procedure as described above with similar modification known to the
one ordinary skilled in the art. The identity of peptide was
confirmed by LCMS (Table 2).
TABLE-US-00005 TABLE 2 Comp LCMS No. Calculated Observed 001 840
840.6 [M].sup.+ 002 882.3 882.6 [M].sup.+ 003 1077.2 1078.8 [M +
H].sup.+ 004 990.3 991.4 [M + H].sup.+ 005 1135.1 1136.1 [M +
H].sup.+ 006 840 841 [M + H].sup.+ 007 868.6 869.6 [M + H].sup.+
008 840 840.4 [M].sup.+ 009 840 840.5 [M].sup.+ 010 896.1 897.1 [M
+ H].sup.+ 011 840 840.4 [M].sup.+ 012 840 840.5 [M].sup.+ 014
896.1 897.1 [M + H].sup.+ 015 840 841 [M + H].sup.+ 016 840 841 [M
+ H].sup.+ 017 840 840.4 [M].sup.+ 018 993.5 994.5 [M + H].sup.+
019 826.1 827.1 [M + H].sup.+ 020 980 980.4 [M].sup.+ 025 925.5
926.7 [M + H].sup.+ 026 925.5 926.7 [M + H].sup.+ 028 897.42 898.4
[M + H].sup.+ 029 810.39 811.3 [M + H].sup.+ 030 696.34 697.7 [M +
H].sup.+ 031 595.30 596.4 [M + H].sup.+
[0209] The remaining compounds of the Table 1 can be prepared by
following similar approach as described above.
[0210] Use of MDA-MB-231 Cells as a Source of PD-L1:
[0211] MDA-MB-231 cells were found to express PD-L1 by RT-PCR and
therefore used as a source of PD-L1 in the assays.
Example 9
The Effect of Peptides on Mouse Splenocyte Proliferation Inhibited
by PDL1/PDL2 or Tumor Cells Expressing PDL
[0212] Requirement:
[0213] Mouse splenocytes harvested from 6-8 weeks old C57 BL6 mice;
RPMI 1640 (GIBCO, Cat #11875); DMEM with high glucose (GIBCO, Cat #
D6429); Fetal Bovine Serum [Hyclone, Cat # SH30071.03]; Pencilin
(10000 unit/ml)-Streptomycin (10,000 .mu.g/ml) Liquid (GIBCO, Cat
#15140-122); MEM Sodium Pyruvate solution 100 mM (100.times.),
Liquid (GIBCO, Cat #11360); Nonessential amino acid (GIBCO, Cat
#11140); L-Glutamine (GIBCO, Cat #25030); Anti-CD3 antibody
(eBiosciences--16-0032); Anti-CD28 antibody
(eBiosciences--16-0281); ACK lysis buffer (1 mL) (GIBCO, Cat
#--A10492); Histopaque (density-1.083 gm/ml) (SIGMA 10831); Trypan
blue solution (SIGMA-T8154); Hemacytometer (Bright line-SIGMA
Z359629); FACS Buffer (PBS/0.1% BSA): Phosphate Buffered Saline
(PBS) pH 7.2 (HiMedia TS1006) with 0.1% Bovine Serum Albumin (BSA)
(SIGMA A7050) and sodium azide (SIGMA 08591); 5 mM stock solution
of CFSE:CFSE stock solution was prepared by diluting lyophilized
CFSE with 1804, of Di methyl Sulfoxide (DMSO C.sub.2H.sub.6SO,
SIGMA-D-5879) and aliquoted in to tubes for further use. Working
concentrations were titrated from 10 .mu.M to 1 .mu.M.
(eBioscience-650850-85); 96-well format ELISA plates (Corning
CLS3390); BD FACS caliber (E6016).
[0214] Protocol
[0215] Splenocyte Preparation:
[0216] Splenocytes harvested in a 50 ml falcon tube by mashing
spleen in a 40 .mu.m cell strainer were further treated with 1 ml
ACK [Ammonium-Chloride-Potassium (K) (chloride)] lysis buffer for 5
mins at RT. After washing with 9 ml of RPMI complete media, cells
re-suspended in 3 ml of 1.times.PBS in a 15 ml tube. 3 ml of
histopaque was added very carefully to the bottom of the tube
without disturbing overlaying splenocyte suspension. Spin the tube
at 800.times.g for 20 mins at RT. Opaque layer of lymphocytes is
collected carefully without disturbing/mixing any of the layers.
Cells washed twice with cold 1.times.PBS followed by total cell
counting using trypan blue exclusion method and used further for
cell based assays.
[0217] CFSE Proliferation Assay:
[0218] CFSE is dye abbreviated as Carboxyfluorescein Diacetate
Succinimidyl Ester that passively diffuses into cells and binds to
intracellular proteins.
[0219] Tumor cells (MDMBA231) are cultured and maintained in high
glucose complete DMEM media. 1.times.10.sup.5 tumor cells were
plated in 96 well plates along with required conc. of PD1 derived
peptide and allowed to adhere at 37.degree. C. for 4 hrs.
1.times.10.sup.6 cells/ml of harvested splenocytes are treated with
5 .mu.M of CFSE in pre warmed 1.times.PBS/0.1% BSA solution for 10
mins at 37.degree. C. Excess CFSE was quenched using 5 volumes of
ice-cold culture media to the cells and incubated on ice for 5
mins. CFSE labeled splenocytes were further given three washes with
ice cold complete DMEM media. CFSE labeled 1.times.10.sup.5
splenocytes added to above wells containing tumors cells and PD1
peptides. Splenocytes were stimulated with anti-CD3 and anti-CD28
antibody (4 .mu.l ml each) and the co-culture was further incubated
for 72 hrs at 37.degree. C. with 5% CO.sub.2. Cells were harvested
and washed thrice with ice cold FACS buffer and % proliferation was
analyzed using a FACS caliber with 488 nm excitation and 521 nm
emission filters. Each experimental condition was carried out in
triplicates and each experiment at least carried out three times. %
splenocyte proliferation was analyzed using cell quest FACS program
and fold induction was calculated by normalizing individual values
to % background proliferation (FIG. 1).
[0220] Fold Induction=% splenocyte proliferation/% background
proliferation
[0221] Stimulated splenocytes: Splenocytes+anti-CD3/CD28
stimulation
[0222] Background proliferation: Splenocytes+anti-CD3/CD28+PDL or
Tumor
[0223] Peptide effect: Splenocytes+anti-CD3/CD28+PDL or
Tumor+Peptide (100 nM)
Example 10
In Vivo Efficacy of Compound 1 on Metastasis of B16F10 Melanoma
[0224] Animals:
[0225] C57/b16J female mice (Aurigene, Bangalore, India) aged 6 to
8 weeks were used for the experiment. Animals were acclimatized for
a week in the experimental room before conducting the
experiment.
[0226] Effect of Compound 001 in B16F10 Metastasis Model
[0227] In the case of metastasis model, 0.1.times.10.sup.6 B16F10
cells were injected to C57/b16J mice through i.v. Compound 001
dissolved in PBS, pH 7.4 was dosed subcutaneously at 5 mg/kg once
daily. Vehicle control group of mice received only saline. Each
group consisted of ten animals. Body weight and clinical signs were
recorded daily. After 14 days of treatment, lung metastasis was
quantitated by counting number of nodules under dissection
microscope. Compound 001 treated at 5 mg/kg showed 64 percent
reduction in metastasis.
Sequence CWU 1
1
331137PRTHomo sapiensPEPTIDEExtracellular domain of Human PD1 1Pro
Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn 1 5 10
15Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
Ala 35 40 45Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe
Arg Val 50 55 60Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val
Val Arg Ala 65 70 75 80Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly
Ala Ile Ser Leu Ala 85 90 95Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val Thr 100 105 110Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro Arg 115 120 125Ser Ala Gly Gln Phe Gln
Thr Leu Val 130 13527PRTHomo sapiensPEPTIDEBC Loop 2Ser Asn Thr Ser
Glu Phe Ser1 537PRTArtificial sequenceSynthetic Peptide 3Ser Asn
Thr Ser Glu Phe Ser1 547PRTArtificial sequenceSynthetic Peptide
4Ser Asn Thr Ser Glu Phe Ser1 557PRTArtificial sequenceSynthetic
Peptide 5Ser Asn Thr Ser Glu Phe Ser1 567PRTArtificial
sequenceSynthetic Peptide 6Ser Asn Thr Ser Glu Phe Ser1
577PRTArtificial sequenceSynthetic Peptide 7Ser Asn Thr Ser Glu Phe
Ser1 587PRTArtificial sequenceSynthetic Peptide 8Ser Asn Thr Ser
Glu Phe Ser1 597PRTArtificial sequenceSynthetic Peptide 9Ser Asn
Thr Ser Glu Phe Ser1 5107PRTArtificial sequenceSynthetic Peptide
10Ser Asn Thr Ser Glu Phe Ser1 5117PRTArtificial sequenceSynthetic
Peptide 11Ser Asn Thr Ser Glu Phe Ser1 5127PRTArtificial
sequenceSynthetic Peptide 12Ser Asn Thr Ser Glu Phe Ser1
5137PRTArtificial sequenceSynthetic Peptide 13Ser Asn Thr Ser Glu
Phe Ser1 5147PRTArtificial sequenceSynthetic Peptide 14Ser Asn Thr
Ser Glu Phe Ser1 5157PRTArtificial sequenceSynthetic Peptide 15Ser
Asn Thr Ser Glu Phe Ser1 5167PRTArtificial sequenceSynthetic
Peptide 16Ser Asn Thr Ser Glu Phe Ser1 5177PRTArtificial
sequenceSynthetic Peptide 17Ser Asn Thr Ser Glu Phe Ser1
5187PRTArtificial sequenceSynthetic Peptide 18Phe Ser Glu Ser Thr
Asn Ser1 5197PRTArtificial sequenceSynthetic Peptide 19Phe Ser Glu
Ser Thr Asn Ser1 5207PRTArtificial sequenceSynthetic Peptide 20Ser
Asn Thr Ser Glu Phe Ser1 5217PRTArtificial sequenceSynthetic
Peptide 21Ser Asn Thr Ser Glu Phe Ser1 5227PRTArtificial
sequenceSynthetic Peptide 22Ser Asn Thr Ser Glu Phe Ser1
5237PRTArtificial sequenceSynthetic Peptide 23Ser Asn Thr Ser Glu
Phe Ser1 5247PRTArtificial sequenceSynthetic Peptide 24Ser Asn Thr
Ser Glu Phe Ser1 5257PRTArtificial sequenceSynthetic Peptide 25Ser
Asn Thr Ser Glu Phe Ser1 5267PRTArtificial sequenceSynthetic
Peptide 26Ser Asn Thr Ser Glu Phe Ser1 5277PRTArtificial
sequenceSynthetic Peptide 27Ser Asn Thr Ser Glu Phe Ser1
5287PRTArtificial sequenceSynthetic Peptide 220> 28Ser Asn Thr
Ser Glu Phe Ser1 5297PRTArtificial sequenceSynthetic Peptide 29Ser
Asn Thr Ser Glu Phe Ser1 5307PRTArtificial sequenceSynthetic
Peptide 30Ser Asn Thr Ser Glu Phe Ser1 5316PRTArtificial
sequenceSynthetic Peptide 31Asn Thr Ser Glu Phe Ser1
5325PRTArtificial sequenceSynthetic Peptide 32Thr Ser Glu Phe Ser1
5334PRTArtificial sequenceSynthetic Peptide 33Ser Glu Phe Ser1
* * * * *